- Email: [email protected]
Deletion size characterization of der(9) deletions in Philadelphia-positive chronic myeloid leukemia
Cancer Genetics and Cytogenetics 170 (2006) 89e92
Lead article
Deletion size characterization of der(9) deletions in Philadelphia-positive chronic myeloid leukemia Nathalie Douet-Guilberta,b, Fre´de´ric Morela,b, Sylvia Quemenera, Aure´lie Maguera, Marie-Jose´e Le Brisb, Patrick Moricec, Christian Berthoud, Marc De Braekeleera,b,* a
Laboratory of Histology, Embryology and Cytogenetics, Faculty of Medicine and Health Sciences, Universite´ de Bretagne Occidentale (UBO), 22 Avenue Camille Desmoulins, CS 93837, F-29238 Brest Cedex 3, France b Department of Cytogenetics, Cytology and Reproductive Biology, CHU Morvan, Avenue Foch, F-29606 Brest, France c Department of Clinical Hematology, CH Yves Le Foll, Avenue de la Beauche´e, F-22023 Saint-Brieuc, France d Department of Clinical Hematology, Institute of Cancerology and Hematology, CHU Morvan, Brest, France Received 30 May 2006; accepted 14 June 2006
Abstract
About 95% of the CML patients with chronic myeloid leukemia (CML) have a Philadelphia chromosome resulting from a reciprocal translocation between bands 9q34 and 22q11.2 that juxtaposes the 30 region of the ABL gene to the 50 region of BCR. Over the past few years, submicroscopic deletions due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint have been found using fluorescence in situ hybridization (FISH). Among 150 CML bone marrow samples analyzed by molecular cytogenetics in our laboratory, 11 had a der(9) deletion detectable by FISH (deletion of the 50 ABL region and 30 BCR region in 10 samples and deletion of the 50 ABL region solely in 1 sample). To delineate the size of the deletions, FISH mapping was performed using 22 bacterial artificial chromosomes (BACs), 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. The deletion size of the 50 ABL region on the der(9) extended from 2 to 5 Mb, the minimal deletion size being localized between BACs RP11-101E3 and RP11-83J21. In two patients, the deletion size of the 30 BCR region was about 500 kb (between RP11-80O7 and RP11-681C06). The poor prognosis associated with these deletions was postulated by several workers to be explained by haploinsufficiency of a tumor suppressor gene. However, in our cases, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment. We think that there could be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising from a neoplastic transformation in a pluripotent stem cell. About 95% of the CML patients have a Philadelphia (Ph) chromosome. The Ph chromosome results from a reciprocal translocation between bands 9q34 and 22q11.2 juxtaposing the 30 region of the ABL (Abelson) oncogene from 9q34 to the 50 of a particular DNA sequence called BCR (breakpoint cluster region) on 22q11. The t(9;22)(q34;q11.2) induces the formation of two new chimeric genes, the 50 ABL-30 BCR gene on the der(9) and the 50 BCR-30 ABL gene on the der(22). The transcript * Corresponding author. Tel.: þ33-298-01-64-76; fax: þ33-298-01-81-89. E-mail address: [email protected] (M. De Braekeleer). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.06.006
of this latter fusion gene leads to the synthesis of a new protein having a strong tyrosine kinase activity and an abnormal signal of cellular proliferation [1,2]. The putative biological or clinical role of the 50 ABL-30 BCR gene expression remains enigmatic, because no 50 abl-30 bcr protein has been found yet. In recent years, submicroscopic deletions of the 50 ABL and 30 BCR regions on the der(9) have been found using fluorescence in situ hybridization (FISH). These deletions are due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint during the formation of the translocation [3e7]. Moreover, our results, as others, suggested that the deletion of the 50 ABL region is associated with a worse prognosis, a shorter chronic phase duration, and shorter overall survival [3,4,6,8,9]. Generally, the 50 ABL region deletion
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patients were resistant to interferon therapy [6,10]. Huntly et al. [11,12] found a lower response rate to imatinib mesylate among patients with a deletion than among nondeletion patients. Quintas-Cardama et al. [13], however, found a similar response rate to imatinib among patients with or without deletion. Some workers postulated that the worse prognosis of deletion patients was associated with loss of tumor suppressor genes [11,14,15]. Only a few studies have tried to determine the deletion size on the der(9) [3,14,16]. In the present study, we characterized the deletion size in 11 CML patients using fluorescence in situ hybridization analysis with bacterial artificial chromosome (BAC) probes and discussed the data available in the literature.
2. Materials and methods 2.1. Patients A total of 150 CML bone marrow samples were analyzed using conventional and molecular cytogenetics. Among those, 11 had a deletion of the 50 ABL region detectable by FISH using LSI BCR-ABL ES (AdGenix, Voisins le Bretonneux, France). Moreover, using BCR-ABL probes (Qbiogene, Illkirch, France) in dual-fusion FISH, we found a double deletion of the 50 ABL region and 30 BCR region on the der(9) among 10 of the 11 patients, the remaining patient having a deletion of the 50 ABL region solely ([6] and unpublished data). 2.2. Bacterial artificial chromosomes To delineate the size of the deletions, FISH mapping was performed with two appropriate sets of BAC clones (Fig. 1). BACs were identified through the Human Genome Browser database of the Genome Bioinformatics Group at the University of California at Santa Cruz (http://genome. ucsc.edu/) and the Ensembl genome data resources of the Sanger Institute Genome Database (http://ensembl.org/). The deletion size was mapped using 22 BACs, 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. Bacterial cultures were prepared from a single colony picked from a selective plate in the presence of chloramphenicol. Plasmids were obtained from bacterial cultures grown in the presence of chloramphenicol (10 mg/mL). After having lysed the bacteria using SDS 1%eNaOH 0.2 mol/L, DNA was purified of RNA, proteins, and other cellular contaminants. Probes were labeled using a Prime-It Fluor fluorescence labeling kit (Stratagene, Amsterdam, Netherlands) with fluorescein isothiocyanate (FITC). 2.3. Fluorescence in situ hybridization All BACs were applied to normal lymphocyte metaphases to verify their chromosomal location. A
9cen 5Mb 4.5Mb 4Mb 3.5Mb 3Mb 2.5Mb 2Mb 1.5Mb 1Mb 0.5Mb
9q34.1
PATIENTS 336P12 494N15 405C6 356B19 203J24 339B21 101E3 65J3 409K20 618A20 83J21
1
2
3
4
5
6
7
8
9 10
11
22q11.2 164N13 0.5Mb 1Mb 1.5Mb 2Mb 2.5Mb 3Mb 3.5Mb 4Mb 4.5Mb 5Mb
80O7 681C06 565B13 50I07 344P23 26A11 669P02 263G19 772E17 155F13
22qter Fig. 1. 50 ABL region deletion size among 11 CML patients, determined using two panels of BACs.
chromosome 9 centromere probe (CEP9, SpectrumOrange) (AdGenix) was used as a control for all FISH techniques. The hybridization procedure and analysis have been described previously [6]. Briefly, before hybridization, DNA slides were immersed in 2 SSCe0.4% NP40 solution for 30 min at 37 C and then were immediately passed through an ethanol series of growing concentration (70, 90, and 100%). The denaturation was performed simultaneously on slides and probes for 1 min at 75 C. The slides were incubated overnight in a dark humidity chamber at 37 C. They were washed for 45 s in 0.4 SSCe0.3% NP40 at 72 C and for 20 s in 2 SSCe0.1% NP40 at room temperature. Finally, they were counterstained with 40 ,6diamidino-2-phenylindole (DAPI). The slides were analyzed using a Zeiss AxioPlan Microscope (Zeiss, Le Pecq, France). Subsequent image acquisition was performed using a charge-coupled device camera with the Isis in situ imaging system (MetaSystems, Altlussheim, Germany).
3. Results 3.1. Deletion of the 50 ABL region on the der(9) The deletion size of the 50 ABL region on the der(9) varied among the 11 patients (Fig. 1), ranging from 2 Mb (4 patients) to 5 Mb (1 patient). Three other patients had a deletion spanning 2.5 Mb, two others 3.5 Mb, and the last one 4.5 Mb. The minimal deletion of the 50 ABL region
N. Douet-Guilbert et al. / Cancer Genetics and Cytogenetics 170 (2006) 89e92
common to all 11 patients localized between BACs RP11101E3 and RP11-83J21 (2 Mb). 3.2. Deletion of the 30 BCR region on the der(9) In 8 of the 11 patients, only BAC RP11-164N13 was deleted. This BAC encompasses the major breakpoint region of the BCR region. For two patients, the deletion was more important, about 500 kb, the breakpoint having been mapped between BACs RP11-80O7 and RP11-681C06. Patient 11 showed no deletion of 30 BCR region (Fig. 1). There was no correlation between the extent of the deletion of the 50 ABL region and that of the 30 BCR region.
4. Discussion Here we have presented the molecular cytogenetic characterization of 11 Ph-positive CML patients showing deletions of the 50 ABL region with or without deletion of the 30 BCR region. The deletion size of the 50 ABL region was relatively important and variable, ranging from 2 to 5 Mb, whereas the deletion size of the 30 BCR region only spanned 0 to 0.5 Mb. Furthermore, the distribution of the breakpoints was not in favor of recombination hot spots within chromosome 9 and chromosome 22 sequences. To our knowledge, few data are available on the der(9) deletions in CML, and all of those studies concluded that the 30 BCR deleted region is shorter than the deleted 50 ABL region [3,14,16]. The del(9)/der(9) ranged from 350 kb to 7 Mb and the del(22)/der(9) from 0 kb to 3.5 Mb [14,16]. Smaller deletions of about 8 to 10 kb were found by Southern blot analysis, but without physiopathologic consequences [17]. Using real-time quantitative polymerase chain reaction (PCR), Kolomietz et al. [18] found microdeletions, undetectable by FISH, extending 120 kb from the 50 end of the ABL gene in the centromeric direction on the der(9). FISH studies found that der(9) deletions were associated with a poor prognosis, including a responsiveness to interferon therapy and a possible negative impact following bone marrow transplantation [8,10]. Moreover, according to Huntly et al. [11], but in contradiction to findings of other workers [13], imatinib mesylate therapy improves, but may not fully reverse, the poor prognosis of patients with deletion of the 50 ABL region. Different molecular mechanisms were postulated to be responsible for the poor prognosis in CML patients with der(9) deletions. First, loss of ABL-BCR expression: this hypothesis is not sufficient because recently, Huntly et al. [4] found that the ABL-BCR expression did not correlate with deletion on der(9) or survival in CML patients. Second, genomic instability: at least three or four breakpoints were necessary for deletion del(9)/der(9) or del(22)/der(9). Thus, a general genetic instability mechanism could be responsible of the poor prognosis. However, Silly et al. [19] and
91
Wada et al. [20], using microsatellite analysis, and more recently Fournier et al. [7], found no evidence of genetic instability in the malignant cells of CML patients with deletions. Third, formation of a novel ABL-BCR fusion gene: variations in the localization of del(9)/der(9) and del(22)/der(9) breakpoints exclude this hypothesis. Fourth, BCR-ABL transcription could be enhanced as a consequence of the aberrant rearrangement on the der(22) generated by the deletions on the der(9). However, Huntly et al. [21], using real time quantitative reverse transcriptase, found that transcript levels were not different between CML patients with or without deletions in the der(9). Huntly et al. [12] suggested that the poor prognosis associated with the deletions could be explained by haploinsufficiency of a tumor suppressor gene. About 50 genes with known function flanking the breakpoint regions on chromosomes 9 and 22 are lost. Among these are several tumor suppressor genes: PRDM12 (PR domain containing 12), ASS (argininosuccinate synthetase), and PTGES (prostaglandin E synthase) on 9q34 and SMARCB1, GSTT1, and GSTT2 (glutathione S-transferases theta 1 and 2) on 22q11. In our patients, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment ([6] and unpublished data). We think that there may be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions. Thus, the loss of a copy of the gene or genes involved could create a haploinsufficiency responsible for the poor response to interferon treatment.
References [1] Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 1985;315: 550e4. [2] Kurzrock R, Shtalrid M, Talpaz M, Kloetzer WS, Gutterman JU. Expression of c-abl in Philadelphia-positive acute myelogenous leukemia. Blood 1987;70:1584e8. [3] Sinclair PB, Nacheva EP, Leversha M, Telford N, Chang J, Reid A, Bench A, Champion K, Huntly B, Green AR. Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia. Blood 2000;95: 738e44. [4] Huntly BJP, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P, Szer J, Prince HM, Turner P, Grace C, Nacheva EP, Green AR. Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. Blood 2001;98:1732e8. [5] Morel F, Ka C, Le Bris MJ, Herry A, Morice P, Bourquard P, Abgrall JF, Berthou C, De Braekeleer M. Deletion of the 50 abl region in Philadelphia chromosome-positive chronic myeloid leukemia. Leukemia 2003;17:473e4. [6] Morel F, Ka C, Le Bris MJ, Herry A, Morice P, Bourquard P, Abgrall JF, Berthou C, De Braekeleer M. Deletion of the 50 abl region in Philadelphia-positive chronic myeloid leukemia: frequency, origin and prognosis. Leuk Lymphoma 2003;44:1333e8.
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[7] Fournier M, Lacrosse S, Jamar M, Bours V, Herens C. Deletions of the 30 BCR and 50 ABL regions in patients with Philadelphia-positive chronic myeloid leukemia: a one-step process occurring in about 10% of the cases without any evidence of genetic instability in the target cells. Cancer Genet Cytogenet 2005;160:184e7. [8] Kolomietz E, Al-Maghrabi J, Brennan S, Karaskova J, Minkin S, Lipton J, Squire JA. Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis. Blood 2001;97:3581e8. [9] Lee DS, Lee YS, Yun YS, Kim YR, Jeong SS, Lee YK, She CJ, Yoon SS, Shin HR, Kim Y, Cho HI. A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression. Genes Chromosomes Cancer 2003;37:291e9. [10] Cohen N, Rozenfeld-Granot G, Hardan I, Brok-Simoni F, Amariglio N, Rechavi G, Trakhtenbrot L. Subgroup of patients with Philadelphia-positive chronic myelogenous leukemia characterized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis. Cancer Genet Cytogenet 2001;128:114e9. [11] Huntly BJP, Guilhot F, Reid AG, Vassiliou G, Hennig E, Franke C, Byrne J, Brizard A, Niederwiesser D, Freeman-Edward J, Cuthbert G, Bown N, Clark RE, Nacheva EP, Green AR, Deininger MWN. Imatinib improves but may not fully reverse the poor prognosis of patients with CML with derivative chromosome 9 deletions. Blood 2003;102:2205e12. [12] Huntly BJP, Bench A, Green AR. Double jeopardy from a single translocation: deletions of the derivative chromosome 9 in chronic myeloid leukemia. Blood 2003;102:1160e8. [13] Quintas-Cardama A, Kantarjian H, Talpaz M, O’Brien S, GarciaManero G, Verstovsek S, Rios MB, Hayes K, Glassman A, Bekele B, Zhou X, Cortes J. Imatinib mesylate therapy may overcome the poor prognostic significance of deletions of derivative chromosome 9 in patients with chronic myelogenous leukemia. Blood 2005;105:2281e6.
[14] Specchia G, Albano F, Anelli L, Storlazzi CT, Zagaria A, Liso A, Pannunzio A, Pastore D, Mestice A, Greco G, Liso V, Rocchi M. Derivative chromosome 9 deletions in chronic myeloid leukemia are associated with loss of tumor suppressor genes. Leuk Lymphoma 2004;45:689e94. [15] Anelli L, Albano F, Zagaria A, Liso A, Roberti MG, Rocchi M, Specchia G. A chronic myelocytic leukemia case bearing deletions on the three chromosomes involved in a variant t(9;22;11). Cancer Genet Cytogenet 2004;148:137e40. [16] Storlazzi CT, Specchia G, Anelli L, Albano F, Pastore D, Zagaria A, Rocchi M, Liso V. Breakpoint characterization of der(9) deletions in chronic myeloid leukemia patients. Genes Chromosomes Cancer 2002;35:271e6. [17] Popenoe DW, Schaefer-Rego K, Mears JG, Bank A, Leibowitz D. Frequent and extensive deletion during the 9,22 translocation in CML. Blood 1986;68:1123e8. [18] Kolomietz E, Marrano P, Yee K, Thai B, Braude I, Kolomietz A, Chun K, Minkin S, Kamel-Reid S, Minden M, Squire JA. Quantitative PCR identifies a minimal deleted region of 120 kb extending from the Philadelphia chromosome ABL translocation breakpoint in chronic myeloid leukemia with poor outcome. Leukemia 2003;17: 1313e23. [19] Silly H, Chase A, Mills KI, Apfelbeck U, Sormann S, Goldman JM, Cross NC. No evidence for microsatellite instability or consistent loss of heterozygosity at selected loci in chronic myeloid leukaemia blast crisis. Leukemia 1994;8:1923e8. [20] Wada C, Shionoya S, Fujino Y, Tokuhiro H, Akahoshi T, Uchida T, Ohtani H. Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia. Blood 1994;83:3449e56. [21] Huntly BJP, Bench AJ, Delabesse E, Reid AG, Li J, Scott MA, Campbell L, Byrne J, Pinto E, Brizard A, Niedermeiser D, Nacheva EP, Guilhot F, Delninger M, Green AR. Derivative chromosome 9 deletions in chronic myeloid leukemia: poor prognosis is not associated with loss of ABL-BCR expression, elevated BCR-ABL levels, or karyotypic instability. Blood 2002;99:4547e53.
Lead article
Deletion size characterization of der(9) deletions in Philadelphia-positive chronic myeloid leukemia Nathalie Douet-Guilberta,b, Fre´de´ric Morela,b, Sylvia Quemenera, Aure´lie Maguera, Marie-Jose´e Le Brisb, Patrick Moricec, Christian Berthoud, Marc De Braekeleera,b,* a
Laboratory of Histology, Embryology and Cytogenetics, Faculty of Medicine and Health Sciences, Universite´ de Bretagne Occidentale (UBO), 22 Avenue Camille Desmoulins, CS 93837, F-29238 Brest Cedex 3, France b Department of Cytogenetics, Cytology and Reproductive Biology, CHU Morvan, Avenue Foch, F-29606 Brest, France c Department of Clinical Hematology, CH Yves Le Foll, Avenue de la Beauche´e, F-22023 Saint-Brieuc, France d Department of Clinical Hematology, Institute of Cancerology and Hematology, CHU Morvan, Brest, France Received 30 May 2006; accepted 14 June 2006
Abstract
About 95% of the CML patients with chronic myeloid leukemia (CML) have a Philadelphia chromosome resulting from a reciprocal translocation between bands 9q34 and 22q11.2 that juxtaposes the 30 region of the ABL gene to the 50 region of BCR. Over the past few years, submicroscopic deletions due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint have been found using fluorescence in situ hybridization (FISH). Among 150 CML bone marrow samples analyzed by molecular cytogenetics in our laboratory, 11 had a der(9) deletion detectable by FISH (deletion of the 50 ABL region and 30 BCR region in 10 samples and deletion of the 50 ABL region solely in 1 sample). To delineate the size of the deletions, FISH mapping was performed using 22 bacterial artificial chromosomes (BACs), 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. The deletion size of the 50 ABL region on the der(9) extended from 2 to 5 Mb, the minimal deletion size being localized between BACs RP11-101E3 and RP11-83J21. In two patients, the deletion size of the 30 BCR region was about 500 kb (between RP11-80O7 and RP11-681C06). The poor prognosis associated with these deletions was postulated by several workers to be explained by haploinsufficiency of a tumor suppressor gene. However, in our cases, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment. We think that there could be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising from a neoplastic transformation in a pluripotent stem cell. About 95% of the CML patients have a Philadelphia (Ph) chromosome. The Ph chromosome results from a reciprocal translocation between bands 9q34 and 22q11.2 juxtaposing the 30 region of the ABL (Abelson) oncogene from 9q34 to the 50 of a particular DNA sequence called BCR (breakpoint cluster region) on 22q11. The t(9;22)(q34;q11.2) induces the formation of two new chimeric genes, the 50 ABL-30 BCR gene on the der(9) and the 50 BCR-30 ABL gene on the der(22). The transcript * Corresponding author. Tel.: þ33-298-01-64-76; fax: þ33-298-01-81-89. E-mail address: [email protected] (M. De Braekeleer). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.06.006
of this latter fusion gene leads to the synthesis of a new protein having a strong tyrosine kinase activity and an abnormal signal of cellular proliferation [1,2]. The putative biological or clinical role of the 50 ABL-30 BCR gene expression remains enigmatic, because no 50 abl-30 bcr protein has been found yet. In recent years, submicroscopic deletions of the 50 ABL and 30 BCR regions on the der(9) have been found using fluorescence in situ hybridization (FISH). These deletions are due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint during the formation of the translocation [3e7]. Moreover, our results, as others, suggested that the deletion of the 50 ABL region is associated with a worse prognosis, a shorter chronic phase duration, and shorter overall survival [3,4,6,8,9]. Generally, the 50 ABL region deletion
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patients were resistant to interferon therapy [6,10]. Huntly et al. [11,12] found a lower response rate to imatinib mesylate among patients with a deletion than among nondeletion patients. Quintas-Cardama et al. [13], however, found a similar response rate to imatinib among patients with or without deletion. Some workers postulated that the worse prognosis of deletion patients was associated with loss of tumor suppressor genes [11,14,15]. Only a few studies have tried to determine the deletion size on the der(9) [3,14,16]. In the present study, we characterized the deletion size in 11 CML patients using fluorescence in situ hybridization analysis with bacterial artificial chromosome (BAC) probes and discussed the data available in the literature.
2. Materials and methods 2.1. Patients A total of 150 CML bone marrow samples were analyzed using conventional and molecular cytogenetics. Among those, 11 had a deletion of the 50 ABL region detectable by FISH using LSI BCR-ABL ES (AdGenix, Voisins le Bretonneux, France). Moreover, using BCR-ABL probes (Qbiogene, Illkirch, France) in dual-fusion FISH, we found a double deletion of the 50 ABL region and 30 BCR region on the der(9) among 10 of the 11 patients, the remaining patient having a deletion of the 50 ABL region solely ([6] and unpublished data). 2.2. Bacterial artificial chromosomes To delineate the size of the deletions, FISH mapping was performed with two appropriate sets of BAC clones (Fig. 1). BACs were identified through the Human Genome Browser database of the Genome Bioinformatics Group at the University of California at Santa Cruz (http://genome. ucsc.edu/) and the Ensembl genome data resources of the Sanger Institute Genome Database (http://ensembl.org/). The deletion size was mapped using 22 BACs, 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. Bacterial cultures were prepared from a single colony picked from a selective plate in the presence of chloramphenicol. Plasmids were obtained from bacterial cultures grown in the presence of chloramphenicol (10 mg/mL). After having lysed the bacteria using SDS 1%eNaOH 0.2 mol/L, DNA was purified of RNA, proteins, and other cellular contaminants. Probes were labeled using a Prime-It Fluor fluorescence labeling kit (Stratagene, Amsterdam, Netherlands) with fluorescein isothiocyanate (FITC). 2.3. Fluorescence in situ hybridization All BACs were applied to normal lymphocyte metaphases to verify their chromosomal location. A
9cen 5Mb 4.5Mb 4Mb 3.5Mb 3Mb 2.5Mb 2Mb 1.5Mb 1Mb 0.5Mb
9q34.1
PATIENTS 336P12 494N15 405C6 356B19 203J24 339B21 101E3 65J3 409K20 618A20 83J21
1
2
3
4
5
6
7
8
9 10
11
22q11.2 164N13 0.5Mb 1Mb 1.5Mb 2Mb 2.5Mb 3Mb 3.5Mb 4Mb 4.5Mb 5Mb
80O7 681C06 565B13 50I07 344P23 26A11 669P02 263G19 772E17 155F13
22qter Fig. 1. 50 ABL region deletion size among 11 CML patients, determined using two panels of BACs.
chromosome 9 centromere probe (CEP9, SpectrumOrange) (AdGenix) was used as a control for all FISH techniques. The hybridization procedure and analysis have been described previously [6]. Briefly, before hybridization, DNA slides were immersed in 2 SSCe0.4% NP40 solution for 30 min at 37 C and then were immediately passed through an ethanol series of growing concentration (70, 90, and 100%). The denaturation was performed simultaneously on slides and probes for 1 min at 75 C. The slides were incubated overnight in a dark humidity chamber at 37 C. They were washed for 45 s in 0.4 SSCe0.3% NP40 at 72 C and for 20 s in 2 SSCe0.1% NP40 at room temperature. Finally, they were counterstained with 40 ,6diamidino-2-phenylindole (DAPI). The slides were analyzed using a Zeiss AxioPlan Microscope (Zeiss, Le Pecq, France). Subsequent image acquisition was performed using a charge-coupled device camera with the Isis in situ imaging system (MetaSystems, Altlussheim, Germany).
3. Results 3.1. Deletion of the 50 ABL region on the der(9) The deletion size of the 50 ABL region on the der(9) varied among the 11 patients (Fig. 1), ranging from 2 Mb (4 patients) to 5 Mb (1 patient). Three other patients had a deletion spanning 2.5 Mb, two others 3.5 Mb, and the last one 4.5 Mb. The minimal deletion of the 50 ABL region
N. Douet-Guilbert et al. / Cancer Genetics and Cytogenetics 170 (2006) 89e92
common to all 11 patients localized between BACs RP11101E3 and RP11-83J21 (2 Mb). 3.2. Deletion of the 30 BCR region on the der(9) In 8 of the 11 patients, only BAC RP11-164N13 was deleted. This BAC encompasses the major breakpoint region of the BCR region. For two patients, the deletion was more important, about 500 kb, the breakpoint having been mapped between BACs RP11-80O7 and RP11-681C06. Patient 11 showed no deletion of 30 BCR region (Fig. 1). There was no correlation between the extent of the deletion of the 50 ABL region and that of the 30 BCR region.
4. Discussion Here we have presented the molecular cytogenetic characterization of 11 Ph-positive CML patients showing deletions of the 50 ABL region with or without deletion of the 30 BCR region. The deletion size of the 50 ABL region was relatively important and variable, ranging from 2 to 5 Mb, whereas the deletion size of the 30 BCR region only spanned 0 to 0.5 Mb. Furthermore, the distribution of the breakpoints was not in favor of recombination hot spots within chromosome 9 and chromosome 22 sequences. To our knowledge, few data are available on the der(9) deletions in CML, and all of those studies concluded that the 30 BCR deleted region is shorter than the deleted 50 ABL region [3,14,16]. The del(9)/der(9) ranged from 350 kb to 7 Mb and the del(22)/der(9) from 0 kb to 3.5 Mb [14,16]. Smaller deletions of about 8 to 10 kb were found by Southern blot analysis, but without physiopathologic consequences [17]. Using real-time quantitative polymerase chain reaction (PCR), Kolomietz et al. [18] found microdeletions, undetectable by FISH, extending 120 kb from the 50 end of the ABL gene in the centromeric direction on the der(9). FISH studies found that der(9) deletions were associated with a poor prognosis, including a responsiveness to interferon therapy and a possible negative impact following bone marrow transplantation [8,10]. Moreover, according to Huntly et al. [11], but in contradiction to findings of other workers [13], imatinib mesylate therapy improves, but may not fully reverse, the poor prognosis of patients with deletion of the 50 ABL region. Different molecular mechanisms were postulated to be responsible for the poor prognosis in CML patients with der(9) deletions. First, loss of ABL-BCR expression: this hypothesis is not sufficient because recently, Huntly et al. [4] found that the ABL-BCR expression did not correlate with deletion on der(9) or survival in CML patients. Second, genomic instability: at least three or four breakpoints were necessary for deletion del(9)/der(9) or del(22)/der(9). Thus, a general genetic instability mechanism could be responsible of the poor prognosis. However, Silly et al. [19] and
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Wada et al. [20], using microsatellite analysis, and more recently Fournier et al. [7], found no evidence of genetic instability in the malignant cells of CML patients with deletions. Third, formation of a novel ABL-BCR fusion gene: variations in the localization of del(9)/der(9) and del(22)/der(9) breakpoints exclude this hypothesis. Fourth, BCR-ABL transcription could be enhanced as a consequence of the aberrant rearrangement on the der(22) generated by the deletions on the der(9). However, Huntly et al. [21], using real time quantitative reverse transcriptase, found that transcript levels were not different between CML patients with or without deletions in the der(9). Huntly et al. [12] suggested that the poor prognosis associated with the deletions could be explained by haploinsufficiency of a tumor suppressor gene. About 50 genes with known function flanking the breakpoint regions on chromosomes 9 and 22 are lost. Among these are several tumor suppressor genes: PRDM12 (PR domain containing 12), ASS (argininosuccinate synthetase), and PTGES (prostaglandin E synthase) on 9q34 and SMARCB1, GSTT1, and GSTT2 (glutathione S-transferases theta 1 and 2) on 22q11. In our patients, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment ([6] and unpublished data). We think that there may be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions. Thus, the loss of a copy of the gene or genes involved could create a haploinsufficiency responsible for the poor response to interferon treatment.
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